Process for the preparation of 4-acetoxy-2α-benzoyloxy-5β, 20-epoxy-1, 7β-10β-trihydroxy-9-oxo-tax-11-en-13α-yl(2R,3S)-3-tert-b utoxy-carbonYlamino-2-hydroxy-3-phenylpropionate trihydrate

ABSTRACT

Process for the preparation of 4-acetoxy-2α-benzoyloxy-5β,20-epoxy-1,7β, 10β-trihydroxy-9-oxo-tax-11-en-13α-yl (2R,3S)-3-tert-butoxycarbonylamino-2-hydroxy-3-phenylpropionate trihydrate, characterized in that the 4-acetoxy-2α-benzoyloxy-5β,20-epoxy-1,7β,10β-trihydroxy-9-oxo-tax-11-en-13α-yl (2R,3S)-3-tert-butoxycarbonylamino-2-hydroxy-3-phenylpropionate is crystallized from a mixture of water and an aliphatic alcohol containing 1 to 3 carbon atoms, and then the product obtained is dried under defined conditions of temperature, pressure and humidity.

This application is a 371 of PCT/FR95/00910 dated Jul. 7, 1995.

The present invention relates to a process for the preparation of4-acetoxy-2α-benzoyloxy-5β,20-epoxy-1,7β,10β-trihydroxy-9-oxo-tax-11-en-13α-yl(2R,3S)-3-tert-butoxycarbonylamino-2-hydroxy-3-phenylpropionatetrihydrate.

4-Acetoxy-2α-benzoyloxy-5β,20-epoxy-1,7β,10β-trihydroxy-9-oxo-tax-11-en-13α-yl(2R,3S)-3-tertbutoxycarbonylamino-2-hydroxy-3-phenylpropionate, whichhas remarkable anticancer and antileukaemic properties, and itspreparation are described in European Patents EP-0,253,738 andEP-0,336,841.

It has been found that4-acetoxy-2α-benzoyloxy-5β,20-epoxy-1,7β,10β-trihydroxy-9-oxo-tax-11-en-13α-yl(2R,3S)-3-tert-butoxycarbonylamino-2-hydroxy-3-phenylpropionatetrihydrate has a substantially greater stability than that of theanhydrous product.

According to the invention, 4-acetoxy-2α-benzoyloxy-5β,20-epoxy-1,7β,10β-trihydroxy-9-oxo-tax-11-en-13α-yl(2R,3S)-3-tert-butoxycarbonylamino-2-hydroxy-3-phenylpropionatetrihydrate can be obtained by crystallization of4-acetoxy-2α-benzoyloxy-5β,20-epoxy-1,7β,10β-trihydroxy-9-oxo-tax-11-en-13α-yl(2R,3S)-3-tert-butoxycarbonylamino-2-hydroxy-3-phenylpropionate from amixture of water and an aliphatic alcohol containing 1 to 3 carbonatoms, followed by drying of the product obtained under definedconditions of temperature, pressure and humidity.

For the implementation of the process according to the invention, it maybe particularly advantageous

to dissolve the 4-acetoxy-2α-benzoyloxy-5β,20-epoxy-1,7β,10β-trihydroxy-9-oxo-tax-11-en-13α-yl(2R,3S)-3-tert-butoxycarbonylamino-2-hydroxy-3-phenylpropionate, firstlypurified by chromatography, in an aliphatic alcohol containing 1 to 3carbon atoms at a temperature preferably of between 40 and 60° C.,

to optionally remove the residual chromatography solvents byco-distillation under reduced pressure, replacing the volume of solventdistilled off with pure alcohol,

to add optionally purified water at the same temperature,

then, after optionally initiating the crystallization and cooling to atemperature close to 0° C., to separate the crystals obtained from4-acetoxy-2α-benzoyloxy-5β,20-epoxy-1,7β,10β-trihydroxy-9-oxo-tax-11-en-13α-yl(2R,3S)-3-tert-butoxycarbonylamino-2-hydroxy-3-phenylpropionatetrihydrate crystals thus obtained, and then to dry them under reducedpressure in a controlled-humidity atmosphere.

Generally, the purified 4-acetoxy-2α-benzoyloxy-5β,20-epoxy-1,7β,10β-trihydroxy-9-oxo-tax-11-en-13α-yl(2R,3S)-3-tert-butoxycarbonylamino-2-hydroxy-3-phenylpropionate isdissolved in an excess of the aliphatic alcohol. Preferably, thequantity of alcohol is between 8 and 12 parts by weight relative to the4-acetoxy-2α-benzoyloxy-5β,20-epoxy-1,7β,10β-trihydroxy-9-oxo-tax-11-en-13α-yl(2R,3S)-3-tert-butoxycarbonylamino-2-hydroxy-3-phenylpropionate used.

Generally, the distillation of the alcohol is performed under reducedpressure at a temperature close to 40° C. until a thick syrup isobtained which is difficult to stir. It may be advantageous to repeatthis operation several times which leads to the removal of the residualsolvents contained in the purified product used.

After completing the removal of the residual solvents, the syrupobtained is taken up in a quantity of alcohol of between 3.5 and 6 partsby weight relative to the 4-acetoxy-2α-benzoyloxy-5β,20-epoxy-1,7β,10β-trihydroxy-9-oxo-tax-11-en-13α-yl(2R,3S)-3-tert-butoxycarbonylamino-2-hydroxy-3-phenylpropionate.

After optionally separating insoluble impurities by filtration, waterwhich is preferably purified is added such that the water/alcohol weightratio is close to 2/1.

The crystallization is initiated and then the mixture is cooled slowlydown to a temperature close to 0° C.

The4-acetoxy-2α-benzoyloxy-5β,20-epoxy-1,7β,10β-trihydroxy-9-oxo-tax-11-en-13α-yl(2R,3S)-3-tert-butoxycarbonylamino-2-hydroxy-3-phenylpropionatetrihydrate which crystallizes is separated, preferably by filtration orcentrifugation, and then dried. The drying is performed under a reducedpressure of between 4 and 7 kPa, at a temperature close to 40° C. in acontrolled humidity atmosphere, the relative humidity being close to80%.

For the implementation of the process, it may be advantageous to performthe crystallization in the presence of ascorbic acid which is addedduring the dissolution of the purified4-acetoxy-2α-benzoyloxy-5β,20-epoxy-1,7β,10β-trihydroxy-9-oxo-tax-11-en-13α-yl(2R,3S)-3-tert-butoxycarbonylamino-2-hydroxy-3-phenylpropionate in thealcohol. It is possible to use up to 1% by weight of ascorbic acid.

For the implementation of the process, it is particularly advantageousto use ethanol as alcohol.

The trihydrate structure of the 4-acetoxy-2α-benzoyloxy-5β,20-epoxy-1,7β,10β-trihydroxy-9-oxo-tax-11-en-13α-yl(2R,3S)-3-tert-butoxycarbonylamino-2-hydroxy-3-phenylpropionate isconfirmed by X-ray diffraction, by thermogravimetric analysis and bydifferential scanning calorimetry.

More particularly, thermogravimetric analysis shows a mass loss between40 and 140° C. of 6.1%, which corresponds to three molecules of waterper one molecule of4-acetoxy-2α-benzoyloxy-5β,20-epoxy-1,7β,10β-trihydroxy-9-oxo-tax-11-en-13α-yl(2R,3S)-3-tert-butoxycarbonylamino-2-hydroxy-3-phenylpropionate.

The method for assaying the bulk water and the water of hydration bydifferential scanning calorimetry shows the absence of bulk water and anendothermic signal at 132.6° C. corresponds to the dissociation of ahydrate.

4-acetoxy-2α-benzoyloxy-5β,20-epoxy-1,7β,10β-trihydroxy-9-oxo-tax-11-en-13α-yl(2R,3S)-3-tertbutoxycarbonylamino-2-hydroxy-3-phenylpropionatetrihydrate exhibits the property of no longer having a hydroscopiccharacter.

Stability studies show that 4-acetoxy-2α-benzoyloxy-5β,20-epoxy-1,7β,10β-trihydroxy-9-oxo-tax-11-en-13α-yl(2R,3S)-3-tert-butoxycarbonylamino-2-hydroxy-3-phenylpropionatetrihydrate is stable at 4° C., 25° C. and 35° C. in an atmosphere with90% relative humidity up to 18 months without modification of itscrystalline form.

Under the same conditions, anhydrous 4-acetoxy-2α-benzoyloxy-5β,20-epoxy-1,7β,10β-trihydroxy-9-oxo-tax-11-en-13α-yl(2R,3S)-3-tert-butoxycarbonylamino-2-hydroxy-3-phenylpropionate, whosecrystalline form is different, changes slowly to the trihydrate form.

The following examples illustrate the present invention.

EXAMPLE 1

303 g of4-acetoxy-2α-benzoyloxy-5β,20-epoxy-1,7β,10β-trihydroxy-9-oxo-tax-11-en-13α-yl(2R,3S)-3-tert-butoxycarbonylamino-2-hydroxy-3-phenylpropionate whosetitre is 92.4% (0.314 mol) and 2.875 kg of absolute ethanol (d=0.79) areintroduced into a reactor protected from light. The mixture is heated at40° C. until there is complete dissolution of the4-acetoxy-2α-benzoyloxy-5β,20-epoxy-1,7β,10β-trihydroxy-9-oxo-tax-11-en-13α-yl(2R,3S)-3-tert-butoxycarbonylamino-2-hydroxy-3-phenylpropionate. Theethanol is then distilled off at a pressure close to 12 kPa until asyrup at the stirring limit is obtained. 0.983 kg of ethanol is added tothe syrup and the distillation is again performed under the sameconditions. 1.257 kg of ethanol are added to the syrup obtained andheated at 50° C. until there is complete dissolution. The mixture isfiltered hot and then 4.39 kg of purified water are added, over 1 hour,while the temperature is maintained at 50° C. After having initiated thecrystallization, the mixture is cooled to 0° C. over 4 hours. Thecrystals are separated by filtration, washed with 0.909 kg and then0.606 kg of an ethanol-water mixture (1-2 by weight) and then dried at38° C. under reduced pressure (5.07 kPa) in an atmosphere at 80%relative humidity for 48 hours. 266.5 g of4-acetoxy-2α-benzoyloxy-5β,20-epoxy-1,7β,10β-trihydroxy-9-oxo-tax-11-en-13α-yl(2R,3S)-3-tert-butoxycarbonylamino-2-hydroxy-3-phenylpropionatetrihydrate are thus obtained whose analysis shows that itshigh-performance liquid chromatography titre is 98.7% (on a dry basis)and that the water content is 6.15%.

EXAMPLE 2

110.0 g of4-acetoxy-2α-benzoyloxy-5β,20-epoxy-1,7β,10β-trihydroxy-9-oxo-tax-11-en-13α-yl(2R,3S)-3-tert-butoxycarbonylamino-2-hydroxy-3-phenylpropionate with atitre of 92.5% and 0.2224 g of ascorbic acid are dissolved, at atemperature close to 35° C., in 1340 cm³ of ethanol. About 70% of theethanol introduced is distilled off under reduced pressure (8 kPa) at atemperature close to 20° C. The mixture [lacuna] heated to 50° C. andthen filtered. The filter is washed with 3 times 70.5 cm³ of ethanol andthen 860.5 cm³ of purified water are added over 15 minutes at 50° C. Themixture is seeded with a few crystals of 4-acetoxy-2α-benzoyloxy-5β,20-epoxy-1,7β,10β-trihydroxy-9-oxo-tax-11-en-13α-yl(2R,3S)-3-tert-butoxycarbonylamino-2-hydroxy-3-phenylpropionatetrihydrate and then stirred for 30 minutes. 860.5 cm³ of purified waterare then added over 3 hours at 50° C. and then cooled over 3 hours downto a temperature close to 0° C. The slurry is then filtered. Thefiltration cake is washed with 330 g of a water-ethanol mixture (2-1 byweight) and then with 220 g of the same mixture and then dried underreduced pressure (5 kPa) at 38° C. under an atmosphere at 80% relativehumidity. 110.2 g of4-acetoxy-2α-benzoyloxy-5β,20-epoxy-1,7β,10β-trihydroxy-9-oxo-tax-11-en-13α-yl(2R,3S)-3-tert-butoxycarbonylamino-2-hydroxy-3-phenylpropionatetrihydrate are thus obtained with a yield of 98%.

We claim:
 1. A process for the preparation of4-acetoxy-2α-benzoyloxy-5β,20-epoxy-1,7β,10β-trihydroxy-9-oxo-tax-11-en-13α-yl(2R,3S)-3-tertbutoxycarbonylamino-2-hydroxy-3-phenylpropionatetrihydrate, comprisingcrystallizing said4-acetoxy-2α-benzoyloxy-5β,20-epoxy-1,7β,10β-trihydroxy-9-oxo-tax-11-en-13α-yl(2R,3S)-3-tert-butoxycarbonylamino-2-hydroxy-3-phenylpropionatetrihydrate from a mixture of water and an aliphatic alcohol containing 1to 3 carbon atoms, and then drying the product obtained under definedconditions of temperature, pressure, and humidity.
 2. The processaccording to claim 1, wherein the water/alcohol weight ratio is about2/1.
 3. The process according to claim 1, wherein said alcohol isethanol.
 4. The process according to claim 1, wherein said drying stepis performed at a temperature of about 40° C., at a pressure of fromabout 4 to about 7 kPa and in an atmosphere whose relative humidity isabout 80%.
 5. The process according to claim 1, wherein saidcrystallizing step is performed in the presence of ascorbic acid.